Methods for reducing the incidence of oxidative stress using human milk oligosaccharides, vitamin c and anti-inflammatory agents

ABSTRACT

Disclosed are methods of reducing the incidence of oxidative stress in infants, toddlers, and children using nutritional compositions including human milk oligosaccharides. The nutritional compositions including the human milk oligosaccharides are effective at reducing inflammation and the incidence of inflammatory diseases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/334,904 filed on Dec. 22, 2011, now U.S. Pat. No. 9,795,623, whichclaims the benefit of U.S. Provisional Application No. 61/428,863 filedon Dec. 31, 2010 and U.S. Provisional Application No. 61/428,865 filedon Dec. 31, 2010, which disclosures are incorporated by reference intheir entirety.

FIELD OF THE DISCLOSURE

The present disclosure relates to human milk oligosaccharides (HMOs) formodulating inflammation in an infant, toddler, or child. Moreparticularly, the present disclosure relates to human milk fortifiers,preterm and term infant formulas and pediatric formulas comprising HMOsthat can reduce inflammation and thereby prevent and/or treatinflammatory conditions and diseases.

BACKGROUND OF THE DISCLOSURE

The inflammatory response is an attempt by the body to restore andmaintain homeostasis after invasion by an infectious agent, antigenchallenge, or physical, chemical or traumatic damage. While theinflammatory response is generally considered a healthy response toinjury, the immune system can present an undesirable physiologicalresponse if it is not appropriately regulated. Specifically, unregulatedoxidation and associated inflammation are major causes of tissue damageand clinically significant disease in preterm and term infants. This isdue in large part to the immaturity in function of the natural immunesystem of infants, and especially preterm infants.

Breastfeeding has been associated with enhanced development and balancedgrowth and maturation of the infant's respiratory, gastrointestinal andimmune systems, thereby providing protection of the infant to infectionand inflammatory diseases. Breast milk appears to contain endogenousantioxidants, such as superoxide dismutase, glutathione peroxidase andcatalase, or other non-enzymatic antioxidants such as glutathione,lactoferrin and polyphenols, in addition to exogenous antioxidants, suchas vitamins A, C, E and selenium. Further, breast milk includes HMOsthat not only act as pathogen receptor analogues, but activate immunefactors by infant intestinal epithelial cells and/or associated immunecell populations. The function of these breast milk components,functioning as antioxidants and as immune modulators, includes not onlythe protection of breast milk lipids by peroxidation, but may alsoassist in the regulation of inflammatory responses to infection or otherinjury.

Not all infants receive human breast milk. Further, no vaccines arecurrently available for the prevention of inflammatory diseases.Therefore, development of safe and efficacious preventative ortherapeutic methods would be beneficial, especially for infants.

It would therefore be desirable to provide nutritional compositions, andsynthetic infant formulas in particular, that can produce nutritionalbenefits including improved immune system growth and development. Itwould additionally be beneficial if the nutritional compositions couldmodulate inflammation and enhance immunity against microbial infections,including bacterial and viral infections, and other inflammatorydiseases.

SUMMARY OF THE DISCLOSURE

The present disclosure is directed to nutritional compositions,including synthetic infant formulas, synthetic pediatric formulas, andsynthetic child formulas, including at least one HMO, alone or incombination with one or more of long chain polyunsaturated fatty acids(LCPUFAs), antioxidants, and/or nucleotides, for reducing inflammationin an infant, toddler, or child, as well as methods of using thecompositions.

One embodiment is a method of reducing the incidence of oxidative stressin an infant, toddler, or child in need thereof. The method comprisesadministering to the infant, toddler, or child a composition comprisingat least one human milk oligosaccharide selected from the groupconsisting of 6′-silayllactose, 2′-fucosyllactose, andlacto-N-neotetraose.

Another embodiment is a method of reducing the incidence of oxidativestress in an infant, toddler, or child in need thereof. The methodcomprises administering to the infant, toddler, or child a compositioncomprising 6′-silayllactose, 2′-fucosyllactose, and lacto-N-neotetraose.

Another embodiment is a method of increasing glutathione levels in abody of an infant, toddler, or child in need thereof. The methodcomprises administering to the infant, toddler, or child a compositioncomprising 6′-silayllactose, 2′-fucosyllactose, and lacto-N-neotetraose.

Another embodiment is a method of reducing the incidence of oxidativestress in an infant, toddler, or child in need thereof. The methodcomprises administering to the infant, toddler, or child a compositioncomprising at least one neutral human milk oligosaccharide and at leastone acidic human milk oligosaccharide.

Another embodiment is a method of increasing glutathione levels in abody of an infant, toddler, or child in need thereof. The methodcomprises administering to the infant, toddler, or child a compositioncomprising at least one neutral human milk oligosaccharide and at leastone acidic human milk oligosaccharide.

It has been discovered that specific HMOs, such as 3′-sialyllactose,6′-sialyllactose and others as noted herein, are highly effective indampening inflammation generally in infants, toddlers, and children, andspecifically in dampening virus-induced inflammation, includingrespiratory syncytial virus, human parainfluenza, and influenza A, ininfants, toddlers, and children by reducing the production of some keycytokines from human immune cells without increasing viral load, whichmay lead to faster recovery from infections. Surprisingly, it wasdetermined that the HMOs demonstrate the desirable dampening effectseven at very low concentrations, including concentrations lower thanthose found in breast milk. Also, it was unexpectedly found that6′-sialyllactose is immunomodulatory even in the absence of a virus, andinduces the production of monocyte-derived cytokines. It has furtherbeen discovered that although biological reactions often occur within a30 to 60 minute period, and thus a 30 to 60 minute incubation isgenerally used for in vitro procedures, a 24 hour pre-treatment of cellsprovides a closer reflection of the daily pre-exposure to HMOs that abreast-fed infant would receive from breast milk.

Additionally, it has been found that fucosyllated HMOs, including3′-fucosyllactose, alone or in combination with sialic acid, are highlyeffective in inhibiting respiratory viruses. Even at low concentrations,the 3′-fucosyllactose and sialic acid are effective.

Moreover, it has been discovered that specific HMOs act in a synergisticmanner against respiratory viruses, including RSV, when combined with along chain polyunsaturated fatty acid and/or a carotenoid. Thesesynergistic actions dampen virus-induced inflammatory cytokines, andspecifically interferon-inducible protein 10 (IP-10). Additionalcomponents including antioxidants, such as vitamin A and vitamin E, ornucleotides, may also be added to the HMO and long chain polyunsaturatedfatty acid and/or carotenoid combinations.

It has further been found that a combination of HMOs includingacidic/sialylated (e.g., 6′-sialyllactose) and/or neutral/fucosylated(e.g., 2′-fucosyllactose) and/or n-acetylglucosylated (e.g., LNnT)prevents the development of necrotizing entercolitis. Also, these HMOshave been found to decrease the oxidative stress and increaseglutathione levels in infants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting H1N1 virus infectivity of MDCK cells in thepresence of various HMOs as tested in Example 37.

FIG. 2 is a graph depicting blood plasma levels of glutathione frompiglets as measured in Example 38.

FIG. 3 is a graph depicting IP-10 levels resulting from administrationof 3′SL and 6′SL as measured in Example 39.

FIG. 4 is a graph depicting IP-10 levels resulting from administrationof 3′SL and 6′SL as measured in Example 39.

FIG. 5 is a graph depicting IP-10 levels resulting from administrationof LNnT as measured in Example 39.

FIG. 6 is a graph depicting IP-10 levels resulting from administrationof LNnT as measured in Example 39.

FIG. 7 is a graph depicting IL-10 levels resulting from administrationof 3′SL and 6′SL as measured in Example 39.

FIG. 8 is a graph depicting IL-10 levels resulting from administrationof 3′SL and 6′SL as measured in Example 39.

FIG. 9 is a graph depicting IL-10 levels resulting from administrationof LNnT as measured in Example 39.

FIG. 10 is a graph depicting IL-10 levels resulting from administrationof LNnT as measured in Example 39.

FIG. 11A and FIG. 11B are graphs depicting chemiluminescence levelsresulting from administration of 2′FL in the presence or absence ofphorbal myristate acetate (PMA) as measured in Example 40.

DETAILED DESCRIPTION OF THE DISCLOSURE

The nutritional compositions and methods described herein utilize HMOsalone or in combination with long chain polyunsaturated fatty acids,and/or antioxidants, and in particular carotenoids, and/or nucleotidesfor controlling and reducing a number of diseases and conditions relatedto inflammation. These and other features of the nutritionalcompositions and methods, as well as some of the many optionalvariations and additions, are described in detail hereafter.

The terms “retort packaging” and “retort sterilizing” are usedinterchangeably herein, and unless otherwise specified, refer to thecommon practice of filling a container, most typically a metal can orother similar package, with a nutritional liquid and then subjecting theliquid-filled package to the necessary heat sterilization step, to forma sterilized, retort packaged, nutritional liquid product.

The term “aseptic packaging” as used herein, unless otherwise specified,refers to the manufacture of a packaged product without reliance uponthe above-described retort packaging step, wherein the nutritionalliquid and package are sterilized separately prior to filling, and thenare combined under sterilized or aseptic processing conditions to form asterilized, aseptically packaged, nutritional liquid product.

The terms “fat” and “oil” as used herein, unless otherwise specified,are used interchangeably to refer to lipid materials derived orprocessed from plants or animals. These terms also include syntheticlipid materials so long as such synthetic materials are suitable fororal administration to humans.

The term “human milk oligosaccharide” or “HMO”, as used herein, unlessotherwise specified, refers generally to a number of complexcarbohydrates found in human breast milk that can be in acidic orneutral form, and to precursors thereof. Exemplary non-limiting humanmilk oligosaccharides include 3′-sialyllactose, 6′-sialyllactose,3′-fucosyllactose, 2′-fucosyllactose, and lacto-N-neotetraose. Exemplaryhuman milk oligosaccharide precursors includes sialic acid and/orfucose.

The term “shelf stable” as used herein, unless otherwise specified,refers to a nutritional product that remains commercially stable afterbeing packaged and then stored at 18-24° C. for at least 3 months,including from about 6 months to about 24 months, and also includingfrom about 12 months to about 18 months.

The terms “nutritional formulation” or “nutritional composition” as usedherein, are used interchangeably and, unless otherwise specified, referto synthetic formulas including nutritional liquids, nutritionalpowders, nutritional solids, nutritional semi-solids, nutritionalsemi-liquids, nutritional supplements, and any other nutritional foodproduct as known in the art. The nutritional powders may bereconstituted to form a nutritional liquid, all of which comprise one ormore of fat, protein and carbohydrate and are suitable for oralconsumption by a human. The terms “nutritional formulation” or“nutritional composition” do not include human breast milk.

The term “nutritional liquid” as used herein, unless otherwisespecified, refers to nutritional products in ready-to-drink liquid form,concentrated form, and nutritional liquids made by reconstituting thenutritional powders described herein prior to use.

The term “nutritional powder” as used herein, unless otherwisespecified, refers to nutritional products in flowable or scoopable formthat can be reconstituted with water or another aqueous liquid prior toconsumption and includes both spraydried and drymixed/dryblendedpowders.

The term “nutritional semi-solid,” as used herein, unless otherwisespecified, refers to nutritional products that are intermediate inproperties, such as rigidity, between solids and liquids. Somesemi-solids examples include puddings, gelatins, and doughs.

The term “nutritional semi-liquid,” as used herein, unless otherwisespecified, refers to nutritional products that are intermediate inproperties, such as flow properties, between liquids and solids. Somesemi-liquids examples include thick shakes and liquid gels.

The term “infant” as used herein, unless otherwise specified, refers toa person 12 months or younger. The term “preterm infant” as used herein,refers to a person born prior to 36 weeks of gestation.

The term “toddler” as used herein, unless otherwise specified, refers toa person greater than one year of age up to three years of age.

The term “child” as used herein, unless otherwise specified, refers to aperson greater than three years of age up to twelve years of age.

The term “newborn” as used herein, unless otherwise specified, refers toa person from birth up to four weeks of age.

The terms “infant formula” or “synthetic infant formula” as used herein,unless otherwise specified, are used interchangeably and refer toliquid, solid, semi-solid, and semi-liquid human milk replacements orsubstitutes that are suitable for consumption by an infant. Thesynthetic formulas include components that are of semi-purified orpurified origin. As used herein, unless otherwise specified, the terms“semi-purified” or “purified” refer to a material that has been preparedby purification of a natural material or by synthesis. The terms “infantformula” or “synthetic infant formula” do not include human breast milk.

The term “synthetic pediatric formula” as used herein, unless otherwisespecified, refers to liquid, solid, semi-solid, and semi-liquid humanmilk replacements or substitutes that are suitable for consumption by aninfant or toddler up to the age of 36 months (3 years). The syntheticformulas include components that are of semi-purified or purifiedorigin. As used herein, unless otherwise specified, the terms“semi-purified” or “purified” refer to a material that has been preparedby purification of a natural material or by synthesis. The term“synthetic pediatric nutritional formula” does not include human breastmilk.

The term “synthetic child formula” as used herein, unless otherwisespecified, refers to liquid, solid, semi-solid, and semi-liquid humanmilk replacements or substitutes that are suitable for consumption by achild up to the age of 12 years. The synthetic formulas includecomponents that are of semi-purified or purified origin. As used herein,unless otherwise specified, the terms “semi-purified” or “purified”refer to a material that has been prepared by purification of a naturalmaterial or by synthesis. The term “synthetic child nutritional formula”does not include human breast milk.

The term “preterm infant formula” as used herein, unless otherwisespecified, refers to liquid and solid nutritional products suitable forconsumption by a preterm infant.

The term “human milk fortifier” as used herein, unless otherwisespecified, refers to liquid and solid nutritional products suitable formixing with breast milk or preterm infant formula or infant formula forconsumption by a preterm or term infant.

The terms “absence of a virus” or “absent a virus” as used herein withrespect to inducing production of monocyte-derived cytokines, unlessotherwise specified, refer to an individual (e.g., an infant) withoutthe virus or having the virus in an amount less than the amount requiredto illicit an immune response; that is, an amount that is less thanrequired for the body's natural immune response to increase theproduction of cytokines and other immune factors.

The terms “inflammatory disease” or “inflammatory condition” as usedherein, unless otherwise specified, refer to any disease, disorder, orcondition characterized by inflammation. The term “infection-mediatedinflammatory disease” as used herein, unless otherwise specified, refersto an inflammatory disease associated or induced by microbial infection,including viral and bacterial infection.

The terms “susceptible” and “at risk” as used herein, unless otherwisespecified, mean having little resistance to a certain condition ordisease, including being genetically predisposed, having a familyhistory of, and/or having symptoms of the condition or disease.

The terms “modulating” or “modulation” or “modulate” as used herein,unless otherwise specified, refer to the targeted movement of a selectedcharacteristic.

The terms “growth of a virus” or “growth of bacteria” as used herein,unless otherwise specified, refer to the production, proliferation, orreplication of a virus or bacteria.

All percentages, parts and ratios as used herein, are by weight of thetotal composition, unless otherwise specified. All such weights, as theypertain to listed ingredients, are based on the active level and,therefore, do not include solvents or by-products that may be includedin commercially available materials, unless otherwise specified.

Numerical ranges as used herein are intended to include every number andsubset of numbers within that range, whether specifically disclosed ornot. Further, these numerical ranges should be construed as providingsupport for a claim directed to any number or subset of numbers in thatrange. For example, a disclosure of from 1 to 10 should be construed assupporting a range of from 2 to 8, from 3 to 7, from 5 to 6, from 1 to9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All references to singular characteristics or limitations of the presentdisclosure shall include the corresponding plural characteristic orlimitation, and vice versa, unless otherwise specified or clearlyimplied to the contrary by the context in which the reference is made.

All combinations of method or process steps as used herein can beperformed in any order, unless otherwise specified or clearly implied tothe contrary by the context in which the referenced combination is made.

The nutritional compositions and methods may comprise, consist of, orconsist essentially of the essential elements of the compositions andmethods as described herein, as well as any additional or optionalelement described herein or otherwise useful in nutritional productapplications.

Product Form

The nutritional compositions of the present disclosure may be formulatedand administered in any known or otherwise suitable oral product form.Any solid, liquid, semi-solid, semi-liquid, or powder product form,including combinations or variations thereof, are suitable for useherein, provided that such forms allow for safe and effective oraldelivery to the individual of the essential ingredients as also definedherein.

The nutritional compositions of the present disclosure include one ormore HMOs as described herein. The compositions may include one or moreHMOs alone or in combination with other immune enhancing factorsincluding, but not limited, to long chain polyunsaturated acids(LCPUFAs), nucleotides, and antioxidants, such as carotenoids andvitamins, as discussed below.

The nutritional compositions may be in any product form comprising theingredients described herein, and which is safe and effective for oraladministration. The nutritional compositions may be formulated toinclude only the ingredients described herein, or may be modified withoptional ingredients to form a number of different product forms.

The nutritional compositions of the present disclosure are desirablyformulated as dietary product forms, which are defined herein as thoseembodiments comprising the ingredients of the present disclosure in aproduct form that then contains at least one of fat, protein, andcarbohydrate, and preferably also contains vitamins, minerals, orcombinations thereof. The nutritional compositions will comprise atleast HMOs, desirably in combination with at least one of protein, fat,vitamins, and minerals, to produce a nutritional composition.

The nutritional compositions may be formulated with sufficient kinds andamounts of nutrients to provide a sole, primary, or supplemental sourceof nutrition, or to provide a specialized nutritional product for use inindividuals afflicted with specific diseases or conditions or with atargeted nutritional benefit as described below.

Specific non-limiting examples of product forms suitable for use withthe HMO-containing compositions as disclosed herein include, forexample, liquid and powdered dietary supplements, liquid and powderedhuman milk fortifiers, liquid and powdered preterm infant formulas,liquid and powdered infant formulas, liquid and powdered elemental andsemi-elemental formulas, liquid and powdered pediatric formulas, liquidand powdered toddler formulas, and liquid and powdered follow-onformulas suitable for use with infants and children.

Nutritional Liquids

Nutritional liquids include both concentrated and ready-to-feednutritional liquids. These nutritional liquids are most typicallyformulated as suspensions or emulsions, although other liquid forms arewithin the scope of the present disclosure.

Nutritional emulsions suitable for use may be aqueous emulsionscomprising proteins, fats, and carbohydrates. These emulsions aregenerally flowable or drinkable liquids at from about 1° C. to about 25°C. and are typically in the form of oil-in-water, water-in-oil, orcomplex aqueous emulsions, although such emulsions are most typically inthe form of oil-in-water emulsions having a continuous aqueous phase anda discontinuous oil phase.

The nutritional emulsions may be and typically are shelf stable. Thenutritional emulsions typically contain up to about 95% by weight ofwater, including from about 50% to about 95%, also including from about60% to about 90%, and also including from about 70% to about 85%, ofwater by weight of the nutritional emulsions. The nutritional emulsionsmay have a variety of product densities, but most typically have adensity greater than about 1.03 g/mL, including greater than about 1.04g/mL, including greater than about 1.055 g/mL, including from about 1.06g/ml to about 1.12 g/mL, and also including from about 1.085 g/ml toabout 1.10 g/mL.

The nutritional emulsions may have a caloric density tailored to thenutritional needs of the ultimate user, although in most instances theemulsions comprise generally at least 19 kcal/fl oz (660 kcal/liter),more typically from about 20 kcal/fl oz (675-680 kcal/liter) to about 25kcal/fl oz (820 kcal/liter), even more typically from about 20 kcal/floz (675-680 kcal/liter) to about 24 kcal/fl oz (800-810 kcal/liter).Generally, the 22-24 kcal/fl oz formulas are more commonly used inpreterm or low birth weight infants, and the 20-21 kcal/fl oz (675-680to 700 kcal/liter) formulas are more often used in term infants. In someembodiments, the emulsion may have a caloric density of from about50-100 kcal/liter to about 660 kcal/liter, including from about 150kcal/liter to about 500 kcal/liter. In some specific embodiments, theemulsion may have a caloric density of 25, or 50, or 75, or 100kcal/liter.

The nutritional emulsion may have a pH ranging from about 3.5 to about8, but are most advantageously in a range of from about 4.5 to about7.5, including from about 5.5 to about 7.3, including from about 6.2 toabout 7.2.

Although the serving size for the nutritional emulsion can varydepending upon a number of variables, a typical serving size isgenerally at least about 1 mL, or even at least about 2 mL, or even atleast about 5 mL, or even at least about 10 mL, or even at least about25 mL, including ranges from about 1 mL to about 300 mL, including fromabout 4 mL to about 250 mL, and including from about 10 mL to about 240mL.

Nutritional Solids

The nutritional solids may be in any solid form but are typically in theform of flowable or substantially flowable particulate compositions, orat least particulate compositions. Particularly suitable nutritionalsolid product forms include spray dried, agglomerated and/or dryblendedpowder compositions. The compositions can easily be scooped and measuredwith a spoon or similar other device, and can easily be reconstituted bythe intended user with a suitable aqueous liquid, typically water, toform a nutritional composition for immediate oral or enteral use. Inthis context, “immediate” use generally means within about 48 hours,most typically within about 24 hours, preferably right afterreconstitution.

The nutritional powders may be reconstituted with water prior to use toa caloric density tailored to the nutritional needs of the ultimateuser, although in most instances the powders are reconstituted withwater to form compositions comprising at least 19 kcal/fl oz (660kcal/liter), more typically from about 20 kcal/fl oz (675-680kcal/liter) to about 25 kcal/fl oz (820 kcal/liter), even more typicallyfrom about 20 kcal/fl oz (675-680 kcal/liter) to about 24 kcal/fl oz(800-810 kcal/liter). Generally, the 22-24 kcal/fl oz formulas are morecommonly used in preterm or low birth weight infants, and the 20-21kcal/fl oz (675-680 to 700 kcal/liter) formulas are more often used interm infants. In some embodiments, the reconstituted powder may have acaloric density of from about 50-100 kcal/liter to about 660 kcal/liter,including from about 150 kcal/liter to about 500 kcal/liter. In somespecific embodiments, the emulsion may have a caloric density of 25, or50, or 75, or 100 kcal/liter.

Human Milk Oligosaccharides (HMOs)

The nutritional compositions of the present disclosure include at leastone HMO, and in many embodiments, a combination of two or more HMOs.

Oligosaccharides are one of the main components of human breast milk,which contains, on average, 10 grams per liter of neutraloligosaccharides and 1 gram per liter of acidic oligosaccharides. Thecomposition of human milk oligosaccharides is very complex and more than200 different oligosaccharide-like structures are known.

The HMOs may be included in the nutritional compositions alone, or insome embodiments, in combination with other immune enhancing factors(e.g., LCPUFAs, antioxidants, nucleotides, etc.) as described herein.The HMO or HMOs may be isolated or enriched from milk(s) secreted bymammals including, but not limited to: human, bovine, ovine, porcine, orcaprine species. The HMOs may also be produced via microbialfermentation, enzymatic processes, chemical synthesis, or combinationsthereof.

Suitable HMOs for use in the nutritional compositions may include acidicoligosaccharides, neutral oligosaccharides, n-acetylglucosylatedoligosaccharides, and HMO precursors. Specific non-limiting examples ofHMOs that may be included individually or in combination in thecompositions of the present disclosure include: sialic acid (i.e., freesialic acid, lipid-bound sialic acid, protein-bound sialic acid);D-glucose (Glc); D-galactose (Gal); N-acetylglucosamine (GlcNAc);L-fucose (Fuc); fucosyl oligosaccharides (i.e., Lacto-N-fucopentaose I;Lacto-N-fucopentaose II; 2′-Fucosyllactose; 3′-Fucosyllactose;Lacto-N-fucopentaose III; Lacto-N-difucohexaose I; andLactodifucotetraose); non-fucosylated, non-sialylated oligosaccharides(i.e., Lacto-N-tetraose and Lacto-N-neotetraose); sialyloligosaccharides (i.e., 3′-Sialyl-3-fucosyllactose;Disialomonofucosyllacto-N-neohexaose;Monofucosylmonosialyllacto-N-octaose (sialyl Lea);Sialyllacto-N-fucohexaose II; Disialyllacto-N-fucopentaose II;Monofucosyldisialyllacto-N-tetraose); and sialyl fucosyloligosaccharides (i.e., 2′-Sialyllactose; 2-Sialyllactosamine;3′-Sialyllactose; 3′-Sialyllactosamine; 6′-Sialyllactose;6′-Sialyllactosamine; Sialyllacto-N-neotetraose c;Monosialyllacto-N-hexaose; Disialyllacto-N-hexaose I;Monosialyllacto-N-neohexaose I; Monosialyllacto-N-neohexaose II;Disialyllacto-N-neohexaose; Disialyllacto-N-tetraose;Disialyllacto-N-hexaose II; Sialyllacto-N-tetraose a;Disialyllacto-N-hexaose I; and Sialyllacto-N-tetraose b). Also usefulare variants in which the glucose (Glc at the reducing end is replacedby N-acetylglucosamine (e.g., 2′-fucosyl-N-acetylglucosamine (2′-FLNac)is such a variant to 2′-fucosyllactose). These HMOs are described morefully in U.S. Patent Application No. 2009/0098240, which is hereinincorporated by reference in its entirety. Other suitable examples ofHMOs that may be included in the compositions of the present disclosureinclude lacto-N-fucopentaose V, lacto-N-hexaose, para-lacto-N-hexaose,lacto-N-neohexaose, para-lacto-N-neohexaose, monofucosyllacto-N-hexaoseII, isomeric fucosylated lacto-N-hexaose (1), isomeric fucosylatedlacto-N-hexaose (3), isomeric fucosylated lacto-N-hexaose (2),difucosyl-para-lacto-N-neohexaose, difucosyl-para-lacto-N-hexaose,difucosyllacto-N-hexaose, lacto-N-neoocataose, para-lacto-N-octanose,iso-lacto-N-octaose, lacto-N-octaose, monofucosyllacto-neoocataose,monofucosyllacto-N-ocataose, difucosyllacto-N-octaose I,difucosyllacto-N-octaose II, difucosyllacto-N-neoocataose II,difucosyllacto-N-neoocataose I, lacto-N-decaose,trifucosyllacto-N-neooctaose, trifucosyllacto-N-octaose,trifucosyl-iso-lacto-N-octaose, lacto-N-difuco-hexaose II,sialyl-lacto-N-tetraose a, sialyl-lacto-N-tetraose b,sialyl-lacto-N-tetraose c, sialyl-fucosyl-lacto-N-tetraose I,sialyl-fucosyl-lacto-N-tetraose II, and disialyl-lacto-N-tetraose, andcombinations thereof. Particularly suitable nutritional compositionsinclude at least one of the following HMOs or HMO precursors: sialicacid (SA); 3′-Sialyllactose (3′SL); 6′-Sialyllactose (6′SL);2′-Fucosyllactose (2′FL); 3′-Fucosyllactose (3′FL); Lacto-N-tetraose andLacto-N-neotetraose (LNnT), and in particular, combinations of 6′SL and3′SL; combinations of 3′FL and SA; combinations of 2′FL and 3′FL;combinations of 2′FL, 3′SL, and 6′SL; combinations of 3′SL, 3′FL, andLNnT; and combinations of 6′SL, 2′FL, and LNnT.

Other exemplary combinations include: SA, 3′SL, 6′SL, 3′FL, 2′FL, andLNnT; 3′SL, 6′SL, 3′FL, 2′FL, and LNnT; SA, 6′SL, 3′FL, 2′FL, and LNnT;SA, 3′SL, 3′FL, 2′FL, and LNnT; SA, 3′SL, 6′SL, 2′FL, and LNnT; SA,3′SL, 6′SL, 3′FL, and LNnT; SA, 3′SL, 6′SL, 3′FL, and 2′FL; SA and 3′SL;SA and 6′SL; SA and 2′FL; SA and LNnT; SA, 3′SL, and 6′SL; SA, 3′SL and3′FL; SA, 3′SL and 2′FL; SA, 3′SL and LNnT; SA, 6′SL and 3′FL; SA, 6′SL,and 2′FL; SA, 6′SL, and LNnT; SA, 3′FL, and 2′FL; SA, 3′FL, and LNnT;SA, 2′FL, and LNnT; SA, 3′SL, 6′SL, and 3′FL; SA, 3′SL, 6′SL and 2′FL;SA, 3′SL, 6′SL, and LNnT; SA, 3′SL, 3′FL, and 2′FL; SA, 3′SL, 3′FL, andLNnT; SA, 3′SL, 2′FL, and LNnT; SA, 6′SL, 3′FL, and 2′FL; SA, 6′SL,2′FL, and LNnT; SA, 6′SL, 3′FL, and LNnT; SA, 3′FL, 2′FL, and LNnT; SA,6′SL, 2′FL, and LNnT; SA, 3′SL, 3′FL, 2′FL, and LNnT; SA, 6′SL, 3′FL,2′FL, and LNnT; SA, 3′SL, 6′SL, 3′FL, and LNnT; SA, 3′SL, 3′FL, 2′FL,and LNnT; SA, 3′SL, 6′SL, 2′FL, and LNnT; 3′SL, 6′SL, 3′FL, and 2′FL;3′SL, 6′SL, 2′FL, and LNnT; 3′SL, 3′FL, 2′FL, and LNnT; 3′SL, 6′SL,3′FL, and LNnT; 3′SL, 6′SL, and 3′FL; 3′SL, 3′FL, and 2′FL; 3′SL, 2′FL,and LNnT; 3′SL, 6′SL, and 2′FL; 3′SL, 6′SL, and LNnT; 3′SL and 3′FL;3′SL and 2′FL; 3′SL and LNnT; 6′SL and 3′FL; 6′SL and 2′FL; 6′SL andLNnT; 6′SL, 3′FL, and LNnT; 6′SL, 3′FL, 2′FL, and LNnT; 3′FL, 2′FL, andLNnT; 3′FL and LNnT; and 2′FL and LNnT.

The HMOs are present in the nutritional compositions in total amounts ofHMO in the composition (mg of HMO per mL of composition) of at leastabout 0.001 mg/mL, including at least about 0.01 mg/mL, including fromabout 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL toabout 20 mg/mL, including from about 0.001 mg/mL to about 10 mg/mL,including from about 0.01 mg/mL to about 10 mg/mL, including from about0.001 mg/mL to about 5 mg/mL, including from about 0.01 mg/mL to about 5mg/mL, including from about 0.001 mg/mL to about 1 mg/mL, including fromabout 0.001 mg/mL to about 0.23 mg/mL, including from about 0.01 mg/mLto about 0.23 mg/mL of total HMO in the nutritional composition.Typically, the amount of HMO in the nutritional composition will dependon the specific HMO or HMOs present and the amounts of other componentsin the nutritional compositions.

In one specific embodiment when the nutritional product is a nutritionalpowder, the total concentration of HMOs in the nutritional powder isfrom about 0.0005% to about 5%, including from about 0.01% to about 1%(by weight of the nutritional powder).

In another specific embodiment, when the nutritional product is aready-to-feed nutritional liquid, the total concentration of HMOs in theready-to-feed nutritional liquid is from about 0.0001% to about 0.50%,including from about 0.001% to about 0.15%, including from about 0.01%to about 0.10%, and further including from about 0.01% to about 0.03%(by weight of the ready-to-feed nutritional liquid).

In another specific embodiment when the nutritional product is aconcentrated nutritional liquid, the total concentration of HMOs in theconcentrated nutritional liquid is from about 0.0002% to about 0.60%,including from about 0.002% to about 0.30%, including from about 0.02%to about 0.20%, and further including from about 0.02% to about 0.06%(by weight of the concentrated nutritional liquid).

In one specific embodiment, the nutritional composition includes aneutral human milk oligosaccharide in an amount of from about 0.001mg/mL to about 20 mg/mL, including from 0.01 mg/mL to about 20 mg/mL,including from about 0.001 mg/mL to less than 2 mg/mL, and includingfrom about 0.01 mg/mL to less than 2 mg/mL.

In some embodiments, the HMOs are used in combination to provide thedesired immune enhancing effect. For example, in one embodiment, thenutritional composition includes 6′SL in combination with 3′SL in atotal amount of HMO of from about 0.001 mg/mL to about 20 mg/mL,including from about 0.01 mg/mL to about 20 mg/mL, including from about0.001 mg/mL to about 0.23 mg/mL, including from about 0.01 mg/mL toabout 0.23 mg/mL, including from about 0.001 mg/mL to less than 0.15mg/mL, and including from 0.01 mg/mL to less than 0.15 mg/mL of thenutritional composition. In another embodiment, the nutritionalcomposition includes 6′SL in combination with 3′SL in a total amount ofHMO of from about 0.001 mg/mL to about 20 mg/mL, including from about0.01 mg/mL to about 20 mg/mL and including greater than 0.65 mg/mL toabout 20 mg/mL.

In one specific embodiment, the nutritional composition includes 6′SL,alone or in combination with other HMOs, in an amount of from about0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about20 mg/mL, including from about 0.001 mg/mL to less than 0.25 mg/mL,including from about 0.01 mg/mL to less than 0.25 mg/mL, including fromgreater than 0.4 mg/mL to about 20 mg/mL, including from about 0.1 mg/mLto about 0.5 mg/mL, and including from about 0.1 mg/mL to about 0.5mg/mL.

In one embodiment, when the nutritional composition includes 6′SL, thetotal amount of HMOs in the nutritional composition includes at leastabout 88% (by total weight HMOs) 6′SL, including from about 88% (bytotal weight HMOs) to about 96% (by total weight HMOs), including fromabout 88% (by total weight HMOs) to about 100% (by total weight HMOs),and including about 100% (by total weight HMOs) 6′SL.

In another embodiment, the nutritional composition includes 3′SL, aloneor in combination with other HMOs, in an amount of from about 0.001mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 20mg/mL, including from about 0.001 mg/mL to less than 0.15 mg/mL,including from about 0.01 mg/mL to less than 0.15 mg/mL, and includingfrom greater than 0.25 mg/mL to about 20 mg/mL.

In one embodiment, when the nutritional composition includes 3′SL, thetotal amount of HMOs in the nutritional composition includes at leastabout 85% (by total weight HMOs) 3′SL, including from about 85% (bytotal weight HMOs) to about 88% (by total weight HMOs), including fromabout 85% (by total weight HMOs) to about 100% (by total weight HMOs),and including about 100% (by total weight HMOs) 3′SL.

In one specific embodiment, the nutritional composition includes LNnT,alone or in combination with other HMOs, in an amount of from about0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about20 mg/mL, including from about 0.001 mg/mL to less than 0.2 mg/mL,including from about 0.01 mg/mL to less than 0.2 mg/mL, and includingfrom greater than 0.32 mg/mL to about 20 mg/mL.

In another specific embodiment, the nutritional composition includes3′FL, alone or in combination with other HMOs, in an amount of fromabout 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL toabout 20 mg/mL, including from about 0.01 mg/mL to less than 1 mg/mL,and including from greater than 1.7 mg/mL to about 20 mg/mL.

In one specific embodiment, the nutritional composition includes 3′FL incombination with SA in a total amount of HMO of from about 0.001 mg/mLto about 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL. Inone embodiment, the nutritional composition includes 3′FL in an amountof from 0.001 mg/mL to less than 1 mg/mL, including from 0.01 mg/mL toless than 1 mg/mL and SA in an amount of about 1 mg/mL.

In another embodiment, the nutritional composition includes 2′FL, aloneor in combination with other HMOs, in an amount of from about 0.001mg/mL to about 20 mg/mL, including from about 0.01 mg/mL to about 20mg/mL, including from about 0.001 mg/mL to less than 2 mg/mL, andincluding from about 0.01 mg/mL to less than 2 mg/mL. In anotherembodiment, the nutritional composition includes 2′FL, alone or incombination with other HMOs, in an amount of from about 0.001 mg/mL toabout 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL andincluding greater than 2.5 mg/mL to about 20 mg/mL.

In one specific embodiment, the nutritional composition includes 2′FL incombination with 3′FL in a total amount of HMO of from 0.001 mg/mL toabout 20 mg/mL, including from about 0.01 mg/mL to about 20 mg/mL.

In yet another embodiment, the nutritional composition includes acombination of 6′ SL, 2′FL, and LNnT in a total amount of HMO of fromabout 0.001 mg/mL to about 20 mg/mL, including from about 0.01 mg/mL toabout 20 mg/mL.

Long Chain Polyunsaturated Fatty Acids (LCPUFAs)

In addition to the HMOs described above, the nutritional products of thepresent disclosure may include LCPUFAs. LCPUFAs are included in thenutritional compositions to provide nutritional support, as well as toreduce oxidative stress and enhance growth and functional development ofthe intestinal epithelium and associated immune cell populations. Insome embodiments, the nutritional composition includes a combination ofone or more HMOs and one or more LCPUFAs such that the compositionprovides a synergistic benefit to the end user, such as a synergisticbenefit in modulating anti-viral immune responses and dampeninginflammation. In some embodiments, the HMO or HMOs used in combinationwith the LCPUFAs to provide the synergistic effect are acidic HMOs.

Exemplary LCPUFAs for use in the nutritional compositions include, forexample, ω-3 LCPUFAs and ω-6 LCPUFAs. Specific LCPUFAs includedocosahexaenoic acid (DHA), eicosapentaenoic acid (EPA),docosapentaenoic acid (DPA), arachidonic acid (ARA), linoleic acid,linolenic acid (alpha linolenic acid) and gamma-linolenic acid derivedfrom oil sources such as plant oils, marine plankton, fungal oils, andfish oils. In one particular embodiment, the LCPUFAs are derived fromfish oils such as menhaden, salmon, anchovy, cod, halibut, tuna, orherring oil. Particularly preferred LCPUFAs for use in the nutritionalcompositions with the HMOs include DHA, ARA, EPA, DPA, and combinationsthereof.

In order to reduce potential side effects of high dosages of LCPUFAs inthe nutritional compositions, the content of LCPUFAs preferably does notexceed 3% by weight of the total fat content, including below 2% byweight of the total fat content, and including below 1% by weight of thetotal fat content in the nutritional composition.

The LCPUFA may be provided as free fatty acids, in triglyceride form, indiglyceride form, in monoglyceride form, in phospholipid form, inesterfied form or as a mixture of one or more of the above, preferablyin triglyceride form.

The nutritional compositions as described herein will typically comprisetotal concentrations of LCPUFA of from about 0.01 mM to about 10 mM andincluding from about 0.01 mM to about 1 mM. Alternatively, thenutritional compositions comprise total concentrations of LCPUFA of fromabout 0.001 g/L to about 1 g/L.

In one embodiment, the nutritional compositions include total long chainω-6 fatty acids in a concentration of from about 100 to about 425 mg/Lor from about 12 to about 53 mg per 100 kcals and/or further includetotal long chain ω-3 fatty acids in a concentration of from about 40 toabout 185 mg/L or from about 5 to about 23 mg per 100 kcals. In onespecific embodiment, the ratio of long chain ω-6 fatty acids to longchain ω-3 fatty acids in the nutritional compositions ranges from about2:1 to about 3:1, preferably about 2.5:1.

In one specific embodiment, the nutritional compositions include DHA ina concentration of from about 0.025 mg/mL to about 0.130 mg/mL or fromabout 3 to about 16 mg per 100 kcals. In another embodiment, thenutritional compositions include ARA in a concentration of from about0.080 mg/mL to about 0.250 mg/mL or from about 10 to about 31 mg per 100kcals. In yet another embodiment, the nutritional compositions includecombinations of DHA and ARA such that the ratio of DHA to ARA rangesfrom about 1:4 to about 1:2.

Antioxidants

Additionally, the nutritional compositions may comprise one or moreantioxidants in combination with the HMOs (and optionally LCPUFAs and/ornucleotides also) to provide nutritional support, as well as to reduceoxidative stress. In some embodiments, the nutritional compositionincludes a combination of HMOs and antioxidants such that thecomposition provides a synergistic benefit to the end user, such as asynergistic benefit in modulating anti-viral immune responses anddampening inflammation. In some embodiments, the HMO or HMOs is used incombination with carotenoids (and specifically lutein, beta-carotene,zeaxanthin and/or lycopene) to provide the synergistic effect.

Any antioxidants suitable for oral administration may be included foruse in the nutritional compositions of the present disclosure,including, for example, vitamin A, vitamin E, vitamin C, retinol,tocopherol, and carotenoids, including lutein, beta-carotene,zeaxanthin, and lycopene, and combinations thereof, for example.

As noted, the antioxidants for use in the nutritional compositions maybe used with the HMOs alone or in combination with HMOs and LCPUFAsand/or nucleotides. In one specific embodiment, the antioxidants for usein the nutritional compositions include carotenoids, and particularly,combinations of the carotenoids lutein, lycopene, zeaxanthin and/orbeta-carotene. Nutritional compositions containing these combinations,as selected and defined herein, can be used to modulate inflammationand/or levels of C-reactive protein in preterm and term infants.

It is generally preferable that the nutritional compositions comprise atleast one of lutein, lycopene, zeaxanthin, and beta-carotene to providea total amount of carotenoid of from about 0.001 μg/mL to about 10μg/mL. More particularly, the nutritional compositions comprise luteinin an amount of from about 0.001 μg/mL to about 10 μg/mL, including fromabout 0.001 μg/mL to about 5 μg/mL, including from about 0.001 μg/mL toabout 0.0190 μg/mL, including from about 0.001 μg/mL to about 0.0140μg/mL, and also including from about 0.044 μg/mL to about 5 μg/mL oflutein. It is also generally preferable that the nutritionalcompositions comprise from about 0.001 μg/mL to about 10 μg/mL,including from about 0.001 μg/mL to about 5 μg/mL, from about 0.001μg/mL to about 0.0130 μg/mL, including from about 0.001 μg/mL to about0.0075 μg/mL, and also including from about 0.0185 μg/mL to about 5μg/mL of lycopene. It is also generally preferable that the nutritionalcompositions comprise from about 1 μg/mL to about 10 μg/mL, includingfrom about 1 μg/mL to about 5 μg/mL, including from about 0.001 μg/mL toabout 0.025 μg/mL, including from about 0.001 μg/mL to about 0.011μg/mL, and also including from about 0.034 μg/mL to about 5 μg/mL ofbeta-carotene. It should be understood that any combination of theseamounts of beta-carotene, lutein, zeaxanthin, and lycopene can beincluded in the nutritional compositions of the present disclosure.Other carotenoids may optionally be included in the nutritionalcompositions as described herein. Any one or all of the carotenoidsincluded in the nutritional compositions described herein may be from anatural source, or artificially synthesized.

Each of the carotenoids in the selected combinations can be obtainedfrom any known or otherwise suitable material source for use innutritional compositions, and each can be provided individually, or alltogether, or in any combination and from any number of sources,including sources such as multivitamin premixes containing othervitamins or minerals in combination with one or more of the carotenoidsas described herein. Non-limiting examples of some suitable sources oflutein, lycopene, beta-carotene, or combinations thereof includeLycoVit® lycopene (available from BASF, Mount Olive, N.J.), Lyc-O-Mato®tomato extract in oil, powder, or bead form (available from LycoRedCorp., Orange, N.J.), beta-carotene, lutein, or lycopene (available fromDSM Nutritional Products, Parsippany, N.J.), FloraGLO® lutein (availablefrom Kemin Health, Des Moines, Iowa), Xangold® Natural Lutein Esters(available from Cognis, Cincinnati, Ohio), and Lucarotin® beta-carotene(available from BASF, Mount Olive, N.J).

Nucleotides

In addition to the HMOs, the nutritional compositions of the presentdisclosure may additionally comprise nucleotides and/or nucleotideprecursors selected from the group consisting of nucleosides, purinebases, pyrimidine bases, ribose and deoxyribose. The nucleotide may bein monophosphate, diphosphate, or triphosphate form. The nucleotide maybe a ribonucleotide or a deoxyribonucleotide. The nucleotides may bemonomeric, dimeric, or polymeric (including RNA and DNA). The nucleotidemay be present in the nutritional composition as a free acid or in theform of a salt, preferably a monosodium salt. In some embodiments, thenutritional composition includes a combination of HMOs and nucleotidessuch that the composition provides a synergistic benefit to the enduser, such as a synergistic benefit in modulating anti-viral immuneresponses and dampening inflammation and/or improving intestinal barrierintegrity.

Incorporation of nucleotides in the nutritional compositions of thepresent disclosure improves intestinal barrier integrity and/ormaturation, which is beneficial to preterm and term infants who haveless developed intestinal flora and hence a slower maturing intestinalbarrier.

Suitable nucleotides and/or nucleosides for use in the nutritionalcompositions include one or more of cytidine 5′-monophosphate, uridine5′-monophosphate, adenosine 5′-monophosphate, guanosine5′-1-monophosphate, and/or inosine 5′-monophosphate, more preferablycytidine 5′-monophosphate, uridine 5′-monophosphate, adenosine5′-monophosphate, guanosine 5′-monophosphate, and inosine5′-monophosphate.

The nucleotides are present in the nutritional products in total amountsof nucleotides of at least about 5 mg/L, including at least about 10mg/L, including from about 10 mg/L to about 200 mg/L, including fromabout 42 mg/L to about 102 mg/L, and including at least about 72 mg/L ofthe nutritional product.

In one specific embodiment when the nutritional composition is anutritional powder, the nucleotide may be present at a level of at leastabout 0.007%, including from about 0.0078% to about 0.1556%, andincluding about 0.056% (by weight of the nutritional powder), or atleast about 0.007 grams, including from about 0.0078 grams to about0.1556 grams, and including about 0.056 grams of nucleotide per 100grams of nutritional powder.

In another specific embodiment, when the nutritional composition is aready-to-feed nutritional liquid, the nucleotide is present at a levelof at least about 0.001%, including from about 0.001% to about 0.0197%,and including about 0.0071% (by weight of the nutritional liquid), or atleast about 0.001 grams, including from about 0.001 grams to about0.0197 grams, and including about 0.0071 grams of nucleotide per 100grams of ready-to-feed nutritional liquid.

In another specific embodiment when the nutritional composition is aconcentrated nutritional liquid, the nucleotide is present at a level ofat least about 0.0019%, including from about 0.0019% to about 0.0382%,and including about 0.0138% (by weight of the nutritional liquid), or atleast about 0.0019 grams, including from about 0.0019 grams to about0.0382 grams, and including about 0.0138 grams of nucleotide per 100grams of concentrated nutritional liquid.

Macronutrients

The nutritional compositions including the HMO or HMOs may be formulatedto include at least one of protein, fat, and carbohydrate. In manyembodiments, the nutritional compositions will include the HMO or HMOswith protein, carbohydrate and fat.

Although total concentrations or amounts of the fat, protein, andcarbohydrates may vary depending upon the product type (i.e., human milkfortifier, preterm infant formula, infant formula, etc.), product form(i.e., nutritional solid, powder, ready-to-feed liquid, or concentratedliquid) and targeted dietary needs of the intended user, suchconcentrations or amounts most typically fall within one of thefollowing embodied ranges, inclusive of any other essential fat,protein, and/or carbohydrate ingredients as described herein.

For the liquid preterm and term infant formulas, carbohydrateconcentrations most typically range from about 5% to about 40%,including from about 7% to about 30%, including from about 10% to about25%, by weight of the preterm or term infant formula; fat concentrationsmost typically range from about 1% to about 30%, including from about 2%to about 15%, and also including from about 3% to about 10%, by weightof the preterm or term infant formula; and protein concentrations mosttypically range from about 0.5% to about 30%, including from about 1% toabout 15%, and also including from about 2% to about 10%, by weight ofthe preterm or term infant formula.

For the liquid human milk fortifiers, carbohydrate concentrations mosttypically range from about 10% to about 75%, including from about 10% toabout 50%, including from about 20% to about 40%, by weight of the humanmilk fortifier; fat concentrations most typically range from about 10%to about 40%, including from about 15% to about 37%, and also includingfrom about 18% to about 30%, by weight of the human milk fortifier; andprotein concentrations most typically range from about 5% to about 40%,including from about 10% to about 30%, and also including from about 15%to about 25%, by weight of the human milk fortifier.

The amount of carbohydrates, fats, and/or proteins in any of the liquidnutritional compositions described herein may also be characterized inaddition to, or in the alternative, as a percentage of total calories inthe liquid nutritional composition as set forth in the following table.These macronutrients for liquid nutritional compositions of the presentdisclosure are most typically formulated within any of the caloricranges (embodiments A-F) described in the following table (eachnumerical value is preceded by the term “about”).

Nutrient % Total Cal. Embodiment A Embodiment B Embodiment CCarbohydrate  0-98  2-96 10-75 Protein  0-98  2-96  5-70 Fat  0-98  2-9620-85 Embodiment D Embodiment E Embodiment F Carbohydrate 30-50 25-5025-50 Protein 15-35 10-30  5-30 Fat 35-55  1-20  2-20

In one specific example, liquid infant formulas (both ready-to-feed andconcentrated liquids) include those embodiments in which the proteincomponent may comprise from about 7.5% to about 25% of the caloriccontent of the formula; the carbohydrate component may comprise fromabout 35% to about 50% of the total caloric content of the infantformula; and the fat component may comprise from about 30% to about 60%of the total caloric content of the infant formula. These ranges areprovided as examples only, and are not intended to be limiting.Additional suitable ranges are noted in the following table (eachnumerical value is preceded by the term “about”).

Nutrient % Total Cal. Embodiment G Embodiment H Embodiment ICarbohydrates: 20-85 30-60 35-55 Fat:  5-70 20-60 25-50 Protein:  2-75 5-50  7-40

When the nutritional product is a powdered preterm or term infantformula, the protein component is present in an amount of from about 5%to about 35%, including from about 8% to about 12%, and including fromabout 10% to about 12% by weight of the preterm or term infant formula;the fat component is present in an amount of from about 10% to about35%, including from about 25% to about 30%, and including from about 26%to about 28% by weight of the preterm or term infant formula; and thecarbohydrate component is present in an amount of from about 30% toabout 85%, including from about 45% to about 60%, including from about50% to about 55% by weight of the preterm or term infant formula.

For powdered human milk fortifiers the protein component is present inan amount of from about 1% to about 55%, including from about 10% toabout 50%, and including from about 10% to about 30% by weight of thehuman milk fortifier; the fat component is present in an amount of fromabout 1% to about 30%, including from about 1% to about 25%, andincluding from about 1% to about 20% by weight of the human milkfortifier; and the carbohydrate component is present in an amount offrom about 15% to about 75%, including from about 15% to about 60%,including from about 20% to about 50% by weight of the human milkfortifier.

The total amount or concentration of fat, carbohydrate, and protein, inthe powdered nutritional compositions of the present disclosure can varyconsiderably depending upon the selected composition and dietary ormedical needs of the intended user. Additional suitable examples ofmacronutrient concentrations are set forth below. In this context, thetotal amount or concentration refers to all fat, carbohydrate, andprotein sources in the powdered product. For powdered nutritionalcompositions, such total amounts or concentrations are most typicallyand preferably formulated within any of the embodied ranges described inthe following table (each numerical value is preceded by the term“about”).

Nutrient % Total Cal. Embodiment J Embodiment K Embodiment LCarbohydrate 1-85 30-60 35-55 Fat 5-70 20-60 25-50 Protein 2-75  5-50 7-40Fat

The nutritional compositions of the present disclosure may, in additionto the LCPUFAs described above, comprise an additional source or sourcesof fat. Suitable additional sources of fat for use herein include anyfat or fat source that is suitable for use in an oral nutritionalproduct and is compatible with the essential elements and features ofsuch products. For example, in one specific embodiment, the additionalfat is derived from short chain fatty acids.

Additional non-limiting examples of suitable fats or sources thereof foruse in the nutritional products described herein include coconut oil,fractionated coconut oil, soybean oil, corn oil, olive oil, saffloweroil, high oleic safflower oil, oleic acids (EMERSOL 6313 OLEIC ACID,Cognis Oleochemicals, Malaysia), MCT oil (medium chain triglycerides),sunflower oil, high oleic sunflower oil, palm and palm kernel oils, palmolein, canola oil, marine oils, fish oils, fungal oils, algae oils,cottonseed oils, and combinations thereof.

Protein

The nutritional compositions of the present disclosure may optionallyfurther comprise protein. Any protein source that is suitable for use inoral nutritional compositions and is compatible with the essentialelements and features of such products is suitable for use in thenutritional compositions.

Non-limiting examples of suitable proteins or sources thereof for use inthe nutritional products include hydrolyzed, partially hydrolyzed ornon-hydrolyzed proteins or protein sources, which may be derived fromany known or otherwise suitable source such as milk (e.g., casein,whey), animal (e.g., meat, fish), cereal (e.g., rice, corn), vegetable(e.g., soy) or combinations thereof. Non-limiting examples of suchproteins include milk protein isolates, milk protein concentrates asdescribed herein, casein protein isolates, extensively hydrolyzedcasein, whey protein, sodium or calcium caseinates, whole cow milk,partially or completely defatted milk, soy protein isolates, soy proteinconcentrates, and so forth. In one specific embodiment, the nutritionalcompositions include a protein source derived from milk proteins ofhuman and/or bovine origin.

Carbohydrate

The nutritional products of the present disclosure may furtheroptionally comprise any carbohydrates that are suitable for use in anoral nutritional product and are compatible with the essential elementsand features of such products.

Non-limiting examples of suitable carbohydrates or sources thereof foruse in the nutritional products described herein may includemaltodextrin, hydrolyzed or modified starch or cornstarch, glucosepolymers, corn syrup, corn syrup solids, rice-derived carbohydrates,pea-derived carbohydrates, potato-derived carbohydrates, tapioca,sucrose, glucose, fructose, lactose, high fructose corn syrup, honey,sugar alcohols (e.g., maltitol, erythritol, sorbitol), artificialsweeteners (e.g., sucralose, acesulfame potassium, stevia) andcombinations thereof. A particularly desirable carbohydrate is a lowdextrose equivalent (DE) maltodextrin.

Other Optional Ingredients

The nutritional compositions of the present disclosure may furthercomprise other optional components that may modify the physical,chemical, aesthetic or processing characteristics of the products orserve as pharmaceutical or additional nutritional components when usedin the targeted population. Many such optional ingredients are known orotherwise suitable for use in medical food or other nutritional productsor pharmaceutical dosage forms and may also be used in the compositionsherein, provided that such optional ingredients are safe for oraladministration and are compatible with the essential and otheringredients in the selected product form.

Non-limiting examples of such optional ingredients includepreservatives, emulsifying agents, buffers, fructooligosaccharides,galactooligosaccharides, polydextrose, and other prebiotics, probiotics,pharmaceutical actives, anti-inflammatory agents, additional nutrientsas described herein, colorants, flavors, thickening agents andstabilizers, emulsifying agents, lubricants, and so forth.

The nutritional compositions may further comprise a sweetening agent,preferably including at least one sugar alcohol such as maltitol,erythritol, sorbitol, xylitol, mannitol, isolmalt, and lactitol, andalso preferably including at least one artificial or high potencysweetener such as acesulfame K, aspartame, sucralose, saccharin, stevia,and tagatose. These sweetening agents, especially as a combination of asugar alcohol and an artificial sweetener, are especially useful informulating liquid beverage embodiments of the present disclosure havinga desirable favor profile. These sweetener combinations are especiallyeffective in masking undesirable flavors sometimes associated with theaddition of vegetable proteins to a liquid beverage. Optional sugaralcohol concentrations in the nutritional product may range from atleast 0.01%, including from about 0.1% to about 10%, and also includingfrom about 1% to about 6%, by weight of the nutritional product.Optional artificial sweetener concentrations may range from about 0.01%,including from about 0.05% to about 5%, also including from about 0.1%to about 1.0%, by weight of the nutritional product.

A flowing agent or anti-caking agent may be included in the nutritionalcompositions as described herein to retard clumping or caking of thepowder over time and to make a powder embodiment flow easily from itscontainer. Any known flowing or anti-caking agents that are known orotherwise suitable for use in a nutritional powder or product form aresuitable for use herein, non-limiting examples of which includetricalcium phosphate, silicates, and combinations thereof. Theconcentration of the flowing agent or anti-caking agent in thenutritional composition varies depending upon the product form, theother selected ingredients, the desired flow properties, and so forth,but most typically range from about 0.1% to about 4%, including fromabout 0.5% to about 2%, by weight of the nutritional composition.

A stabilizer may also be included in the nutritional compositions. Anystabilizer that is known or otherwise suitable for use in a nutritionalcomposition is also suitable for use herein, some non-limiting examplesof which include gums such as xanthan gum. The stabilizer may representfrom about 0.1% to about 5.0%, including from about 0.5% to about 3%,including from about 0.7% to about 1.5%, by weight of the nutritionalcomposition.

The nutritional compositions may further comprise any of a variety ofother vitamins or related nutrients, non-limiting examples of whichinclude vitamin A, vitamin D, vitamin E, vitamin K, thiamine,riboflavin, pyridoxine, vitamin B₁₂, carotenoids (e.g., beta-carotene,zeaxanthin, lutein, lycopene), niacin, folic acid, pantothenic acid,biotin, vitamin C, choline, inositol, salts and derivatives thereof, andcombinations thereof.

The nutritional compositions may further comprise any of a variety ofother additional minerals, non-limiting examples of which includecalcium, phosphorus, magnesium, iron, zinc, manganese, copper, sodium,potassium, molybdenum, chromium, chloride, and combinations thereof.

Methods of Manufacture

The nutritional compositions of the present disclosure may be preparedby any known or otherwise effective manufacturing technique forpreparing the selected product solid or liquid form. Many suchtechniques are known for any given product form such as nutritionalliquids or powders and can easily be applied by one of ordinary skill inthe art to the nutritional compositions described herein.

The nutritional compositions of the present disclosure can therefore beprepared by any of a variety of known or otherwise effective formulationor manufacturing methods. In one suitable manufacturing process, forexample, at least three separate slurries are prepared, including aprotein-in-fat (PIF) slurry, a carbohydrate-mineral (CHO-MIN) slurry,and a protein-in-water (PIW) slurry. The PIF slurry is formed by heatingand mixing the oil (e.g., canola oil, corn oil, etc.) and then adding anemulsifier (e.g., lecithin), fat soluble vitamins, and a portion of thetotal protein (e.g., milk protein concentrate, etc.) with continued heatand agitation. The CHO-MIN slurry is formed by adding with heatedagitation to water: minerals (e.g., potassium citrate, dipotassiumphosphate, sodium citrate, etc.), trace and ultra trace minerals (TM/UTMpremix), thickening or suspending agents (e.g. avicel, gellan,carrageenan). The resulting CHO-MIN slurry is held for 10 minutes withcontinued heat and agitation before adding additional minerals (e.g.,potassium chloride, magnesium carbonate, potassium iodide, etc.), and/orcarbohydrates (e.g., HMOs, fructooligosaccharide, sucrose, corn syrup,etc.). The PIW slurry is then formed by mixing with heat and agitationthe remaining protein, if any.

The resulting slurries are then blended together with heated agitationand the pH adjusted to 6.6-7.0, after which the composition is subjectedto high-temperature short-time (HTST) processing during which thecomposition is heat treated, emulsified and homogenized, and thenallowed to cool. Water soluble vitamins and ascorbic acid are added, thepH is adjusted to the desired range if necessary, flavors are added, andwater is added to achieve the desired total solid level. The compositionis then aseptically packaged to form an aseptically packaged nutritionalemulsion. This emulsion can then be further diluted, heat-treated, andpackaged to form a ready-to-feed or concentrated liquid, or it can beheat-treated and subsequently processed and packaged as areconstitutable powder, e.g., spray dried, drymixed, agglomerated.

The nutritional solid, such as a spray dried nutritional powder ordrymixed nutritional powder, may be prepared by any collection of knownor otherwise effective technique, suitable for making and formulating anutritional powder.

For example, when the nutritional powder is a spray dried nutritionalpowder, the spray drying step may likewise include any spray dryingtechnique that is known for or otherwise suitable for use in theproduction of nutritional powders. Many different spray drying methodsand techniques are known for use in the nutrition field, all of whichare suitable for use in the manufacture of the spray dried nutritionalpowders herein.

One method of preparing the spray dried nutritional powder comprisesforming and homogenizing an aqueous slurry or liquid comprisingpredigested fat, and optionally protein, carbohydrate, and other sourcesof fat, and then spray drying the slurry or liquid to produce a spraydried nutritional powder. The method may further comprise the step ofspray drying, drymixing, or otherwise adding additional nutritionalingredients, including any one or more of the ingredients describedherein, to the spray dried nutritional powder.

Other suitable methods for making nutritional products are described,for example, in U.S. Pat. No. 6,365,218 (Borschel, et al.), U.S. Pat.No. 6,589,576 (Borschel, et al.), U.S. Pat. No. 6,306,908 (Carlson, etal.), U.S. Patent Application 20030118703 A1 (Nguyen, et al.), whichdescriptions are incorporated herein by reference to the extent thatthey are consistent herewith.

Methods of Use

The nutritional compositions as described herein can be used to addressone or more of the diseases or conditions discussed herein, or can beused to provide one or more of the benefits described herein, to preterminfants, infants, toddlers, and children. The preterm infant, infant,toddler, or child utilizing the nutritional compositions describedherein may actually have or be afflicted with the disease or conditiondescribed, or may be susceptible to, or at risk of, getting the diseaseor condition (that is, may not actually yet have the disease orcondition, but is at elevated risk as compared to the general populationfor getting it due to certain conditions, family history, etc.) Whetherthe preterm infant, infant, toddler, or child actually has the diseaseor condition, or is at risk or susceptible to the disease or condition,the preterm infant, infant, toddler, or child is classified herein as“in need of” assistance in dealing with and combating the disease orcondition. For example, the preterm infant, infant, toddler, or childmay actually have respiratory inflammation or may be at risk of gettingrespiratory inflammation (susceptible to getting respiratoryinflammation) due to family history or other medical conditions, forexample. Whether the preterm infant, infant, toddler, or child actuallyhas the disease or condition, or is only at risk or susceptible togetting the disease or condition, it is within the scope of the presentdisclosure to assist the preterm infant, infant, toddler, or child withthe nutritional compositions described herein.

Based on the foregoing, because some of the method embodiments of thepresent disclosure are directed to specific subsets or subclasses ofidentified individuals (that is, the subset or subclass of individuals“in need” of assistance in addressing one or more specific diseases orspecific conditions noted herein), not all preterm infants, infants,toddlers, and children will fall within the subset or subclass ofpreterm infants, infants, toddlers, and children as described herein forcertain diseases or conditions.

The nutritional compositions as described herein comprise HMOs, alone orin combination with one or more additional components, to provide anutritional source for reducing inflammation, such as respiratoryinflammation (e.g., respiratory syncytial virus-induced inflammation),enteric inflammation, and nasopharyngeal inflammation. The nutritionalcompositions of the present disclosure comprising HMOs may also provideoptimal development and balanced growth and maturation of the infant'sgastrointestinal and immune systems, thereby enhancing the infant'sability to resist microbial infection and modulate inflammatoryresponses to infection (e.g., increased phagocytosis and increasedproduction of reactive oxidative species).

The nutritional compositions also provide growth and maturation of theintestinal epithelial cells in an infant. In one specific embodiment,the administration of the nutritional compositions of the presentdisclosure including HMOs and nucleotides can further activate immuneactivity in or by the intestinal epithelial cells in a newborn.

Further, the use of HMOs in nutritional compositions can reduce thegrowth of respiratory viruses (e.g., RSV, human parainfluenza virus type2, and influenza A virus), and thus, reduce viral-induced upperrespiratory infections. As such, by utilizing HMOs, alone or incombination with other immune enhancing factors, in a nutritionalproduct, such as an infant formula, it is now possible to provideinfants with an alternative, or supplement, to breast milk that moreclosely mimics the benefits thereof.

Along with improved growth and maturation of the infant's immune systemas described above, the use of the nutritional compositions of thepresent disclosure also functions as an immune modulator, therebyreducing inflammation induced by infection in infants, toddlers, andchildren such as respiratory virus-induced infection, and particularly,RSV-induced inflammation, and other infection-mediated inflammatorydiseases.

The addition of HMOs can further increase glutathione levels in the bodyand blood of an infant, and in specific embodiments, of a preterminfant.

When used in combination with LCPUFAs and/or antioxidants, andparticularly, with carotenoids, the HMOs can reduce oxidative stress,which is a metabolic condition in which there is an increased productionand accumulation of oxidized biomolecules such as lipid peroxides andtheir catabolites, protein carbonyls, and oxidatively damaged DNA. Theoutcomes of oxidative stress range from unwanted changes in metabolismto inflammation and cell and tissue death. Accordingly, by reducing theincidence of unregulated inflammation and oxidation in the infant,damage to the tissue lining and cell death is reduced, further reducingthe incidence of inflammatory diseases, such as necrotizingenterocolitis (NEC).

In addition to the benefits discussed above, it has been discovered thatnutritional products including HMOs can modulate production ofmonocyte-derived cytokines in the infant, even in the absence of avirus. This production results in improved immunity to further preventmicrobial infection and reduce the growth of viruses. In one specificembodiment, monocyte-derived cytokines produced by administration of thenutritional compositions of the present disclosure include, for example,interleukin-10, interleukin-8, interleukin-1α, interleukin-1β,interleukin-1ra, and combinations thereof.

Another benefit of utilizing HMOs in nutritional compositions is that ithas been discovered that HMOs modulate the production of IP-10, which isa chemokine that plays an important role in the inflammatory response toviral infection. Specifically, a positive correlation exists between RSVclinical infection severity in children and serum IP-10 levels.Accordingly, a decrease in IP-10 signals a decrease in severity of RSVinfection. In one specific embodiment, IP-10 production is reduced tothe level found in uninfected controls.

Along with reducing IP-10, HMOs have been found to reduceplatelet-neutrophil complex (PNC) formation, which is present in humanblood and consists of up to 25% of unstimulated neutrophils. As PNCs arepresent in aggregates, they have a greater capacity to initiateinflammatory processes and can increase the production of reactiveoxidative species. Accordingly, a decrease in PNC formation can lead toreduced oxidative stress and inflammation in the infant.

EXAMPLES

The following examples illustrate specific embodiments and/or featuresof the nutritional compositions of the present disclosure. The examplesare given solely for the purpose of illustration and are not to beconstrued as limitations of the present disclosure, as many variationsthereof are possible without departing from the spirit and scope of thedisclosure. All exemplified amounts are weight percentages based uponthe total weight of the composition, unless otherwise specified.

The exemplified compositions are shelf stable nutritional compositionsprepared in accordance with the manufacturing methods described herein,such that each exemplified composition, unless otherwise specified,includes an aseptically processed embodiment and a retort packagedembodiment.

Examples 1-5

Examples 1-5 illustrate ready-to-feed nutritional emulsions of thepresent disclosure, the ingredients of which are listed in the tablebelow. All ingredient amounts are listed as kilogram per 1000 kilogrambatch of product, unless otherwise specified.

Ingredient Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 Water Q.S. Q.S. Q.S. Q.S. Q.S.Condensed Skim Milk 86.64 86.64 86.64 86.64 86.64 Lactose 54.80 54.8054.80 54.80 54.80 High oleic safflower oil 14.10 14.10 14.10 14.10 14.10Soybean oil 10.6 10.6 10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.110.1 3′sialylallactose (3′SL) 0.0948 0.090 0.085 9.479 9.005Galactooligosaccharides (GOS) 8.63 8.63 8.63 8.63 8.63 Whey proteinconcentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate 478.9 g 478.9 g478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g 448.28 g 448.28 g448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g355.74 g Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil368.01 g 368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride premix293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium chloride 226.45 g226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g445.94 g 445.94 g 445.94 g Vitamin mineral premix 142.88 g 142.88 g142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium chloride55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g 58.0 g 58.0 g58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g 53.9 g 53.9 gVitamin A, D₃, E, K₁ premix 47.4 g 47.4 g 47.4 g 47.4 g 47.4 g Citricacid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 g Mixed carotenoid premix26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN AN AN ANL-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalcium phosphate15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium phosphate monobasic13.67 g 13.67 g 13.67 g 13.67 g 13.67 g Riboflavin 2.42 g 2.42 g 2.42 g2.42 g 2.42 g Potassium hydroxide AN AN AN AN AN AN = as needed

Examples 6-10

Examples 6-10 illustrate ready-to-feed nutritional emulsions of thepresent disclosure, the ingredients of which are listed in the tablebelow. All ingredient amounts are listed as kilogram per 1000 kilogrambatch of product, unless otherwise specified.

Ingredient Ex. 6 Ex. 7 Ex. 8 Ex. 9 Ex. 10 Water Q.S. Q.S. Q.S. Q.S. Q.S.Condensed Skim Milk 86.64 86.64 86.64 86.64 86.64 Lactose 54.80 54.8054.80 54.80 54.80 High oleic safflower oil 14.10 14.10 14.10 14.10 14.10Soybean oil 10.6 10.6 10.6 10.6 10.6 Coconut oil 10.1 10.1 10.1 10.110.1 6′sialylallactose (6′SL) 0.0948 0.0901 0.0853 9.479 9.0047Galactooligosaccharides (GOS) 8.63 8.63 8.63 8.63 8.63 Whey proteinconcentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate 478.9 g 478.9 g478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g 448.28 g 448.28 g448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g 355.74 g 355.74 g355.74 g Stabilizer 355.74 g 355.74 g 355.74 g 355.74 g 355.74 g ARA oil368.01 g 368.01 g 368.01 g 368.01 g 368.01 g Nucleotide/chloride premix293.26 g 293.26 g 293.26 g 293.26 g 293.26 g Potassium chloride 226.45 g226.45 g 226.45 g 226.45 g 226.45 g Ascorbic acid 445.94 g 445.94 g445.94 g 445.94 g 445.94 g Vitamin mineral premix 142.88 g 142.88 g142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g 137.8 g 137.8 g 137.8g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g 180.0 g Magnesium chloride55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferrous sulfate 58.0 g 58.0 g 58.0 g58.0 g 58.0 g Choline chloride 53.9 g 53.9 g 53.9 g 53.9 g 53.9 gVitamin A, D₃, E, K₁ premix 47.40 g 47.40 g 47.40 g 47.40 g 47.40 gCitric acid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 g Mixed carotenoidpremix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodium chloride AN AN ANAN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 g Tricalciumphosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassium phosphatemonobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g Riboflavin 2.42 g 2.42g 2.42 g 2.42 g 2.42 g Potassium hydroxide AN AN AN AN AN AN = as needed

Examples 11-15

Examples 11-15 illustrate concentrated liquid emulsions of the presentdisclosure, the ingredients of which are listed in the table below. Allingredient amounts are listed as kilogram per 1000 kilogram batch ofproduct, unless otherwise specified.

Ingredient Ex. 11 Ex. 12 Ex. 13 Ex. 14 Ex. 15 Water Q.S. Q.S. Q.S. Q.S.Q.S. Condensed Skim Milk 157.67 157.67 157.67 157.67 157.67 Lactose108.66 108.66 108.66 108.66 108.66 High oleic safflower oil 26.82 26.8226.82 26.82 26.82 Soybean oil 20.16 20.16 20.16 20.16 20.16 Coconut oil19.24 19.24 19.24 19.24 19.24 3′sialylallactose (3′SL) 0.1896 0.18020.1706 18.958 18.009 Galactooligosaccharides (GOS) 17.67 17.67 17.6717.67 17.67 Whey protein concentrate 12.20 12.20 12.20 12.20 12.20Potassium citrate 1.277 1.277 1.277 1.277 1.277 Calcium carbonate 996.1g 996.1 g 996.1 g 996.1 g 996.1 g Soy lecithin 685.0 g 685.0 g 685.0 g685.0 g 685.0 g Monoglycerides 685.0 g 685.0 g 685.0 g 685.0 g 685.0 gARA oil 684.2 g 684.2 g 684.2 g 684.2 g 684.2 g Nucleotide/chloridepremix 568.9 g 568.9 g 568.9 g 568.9 g 568.9 g Potassium chloride 429.7g 429.7 g 429.7 g 429.7 g 429.7 g Ascorbic acid 293.8 g 293.8 g 293.8 g293.8 g 293.8 g Vitamin mineral premix 276.9 g 276.9 g 276.9 g 276.9 g276.9 g DHA oil 256.1 g 256.1 g 256.1 g 256.1 g 256.1 g Carrageenan200.0 g 200.0 g 200.0 g 200.0 g 200.0 g Magnesium chloride 173.3 g 173.3g 173.3 g 173.3 g 173.3 g Ferrous sulfate 112.7 g 112.7 g 112.7 g 112.7g 112.7 g Choline chloride 104.8 g 104.8 g 104.8 g 104.8 g 104.8 gVitamin A, D₃, E, K₁ premix 86.90 g 86.90 g 86.90 g 86.90 g 86.90 gCitric acid 57.50 g 57.50 g 57.50 g 57.50 g 57.50 g Mixed carotenoidpremix 41.90 g 41.90 g 41.90 g 41.90 g 41.90 g Sodium chloride 23.50 g23.50 g 23.50 g 23.50 g 23.50 g L-carnitine 6.40 g 6.40 g 6.40 g 6.40 g6.40 g Tricalcium phosphate AN AN AN AN AN Potassium phosphate monobasicAN AN AN AN AN Potassium hydroxide AN AN AN AN AN AN = as needed

Examples 16-20

Examples 16-20 illustrate ready-to-feed nutritional emulsions of thepresent disclosure, the ingredients of which are listed in the tablebelow. All ingredient amounts are listed as kilogram per 1000 kilogrambatch of product, unless otherwise specified.

Ingredient Ex. 16 Ex. 17 Ex. 18 Ex. 19 Ex. 20 Water Q.S. Q.S. Q.S. Q.S.Q.S. Condensed Skim Milk 86.64 86.64 86.64 86.64 86.64 Lactose 54.8054.80 54.80 54.80 54.80 High oleic safflower oil 14.10 14.10 14.10 14.1014.10 Soybean oil 10.6 10.6 10.6 10.6 10.6 Coconut oil 10.1 10.1 10.110.1 10.1 HMO Mixture 0.0948 0.0901 0.0853 9.479 9.0047 6′sialyllactose(6′SL) 0.0316 0.0300 0.0284 3.159 3.002 2′fucosyllactose (2′FL) 0.03160.0300 0.0284 3.159 3.002 Lacto-N-neotetraose (LNnT) 0.0316 0.03000.0284 3.159 3.002 Galactooligosaccharides (GOS) 8.63 8.63 8.63 8.638.63 Whey protein concentrate 6.40 6.40 6.40 6.40 6.40 Potassium citrate478.9 g 478.9 g 478.9 g 478.9 g 478.9 g Calcium carbonate 448.28 g448.28 g 448.28 g 448.28 g 448.28 g Soy lecithin 355.74 g 355.74 g355.74 g 355.74 g 355.74 g Stabilizer 355.74 g 355.74 g 355.74 g 355.74g 355.74 g ARA oil 368.01 g 368.01 g 368.01 g 368.01 g 368.01 gNucleotide/chloride premix 293.26 g 293.26 g 293.26 g 293.26 g 293.26 gPotassium chloride 226.45 g 226.45 g 226.45 g 226.45 g 226.45 g Ascorbicacid 445.94 g 445.94 g 445.94 g 445.94 g 445.94 g Vitamin mineral premix142.88 g 142.88 g 142.88 g 142.88 g 142.88 g DHA oil 137.8 g 137.8 g137.8 g 137.8 g 137.8 g Carrageenan 180.0 g 180.0 g 180.0 g 180.0 g180.0 g Magnesium chloride 55.0 g 55.0 g 55.0 g 55.0 g 55.0 g Ferroussulfate 58.0 g 58.0 g 58.0 g 58.0 g 58.0 g Choline chloride 53.9 g 53.9g 53.9 g 53.9 g 53.9 g Vitamin A, D₃, E, K₁ premix 47.40 g 47.40 g 47.40g 47.40 g 47.40 g Citric acid 29.77 g 29.77 g 29.77 g 29.77 g 29.77 gMixed carotenoid premix 26.40 g 26.40 g 26.40 g 26.40 g 26.40 g Sodiumchloride AN AN AN AN AN L-carnitine 3.31 g 3.31 g 3.31 g 3.31 g 3.31 gTricalcium phosphate 15.65 g 15.65 g 15.65 g 15.65 g 15.65 g Potassiumphosphate monobasic 13.67 g 13.67 g 13.67 g 13.67 g 13.67 g Riboflavin2.42 g 2.42 g 2.42 g 2.42 g 2.42 g Potassium hydroxide AN AN AN AN AN AN= as needed

Examples 21-25

Examples 21-25 illustrate concentrated liquid emulsions of the presentdisclosure, the ingredients of which are listed in the table below. Allingredient amounts are listed as kilogram per 1000 kilogram batch ofproduct, unless otherwise specified.

Ingredient Ex. 21 Ex. 22 Ex. 23 Ex. 24 Ex. 25 Water Q.S. Q.S. Q.S. Q.S.Q.S. Condensed Skim Milk 157.67 157.67 157.67 157.67 157.67 Lactose108.66 108.66 108.66 108.66 108.66 High oleic safflower oil 26.82 26.8226.82 26.82 26.82 Soybean oil 20.16 20.16 20.16 20.16 20.16 Coconut oil19.24 19.24 19.24 19.24 19.24 HMO Mixture 18.957 18.009 17.061 19.90520.853 6′sialyllactose (6′SL) 6.319 6.003 5.687 6.635 6.9512′fucosyllactose (2′FL) 6.319 6.003 5.687 6.635 6.951Lacto-N-neotetraose (LNnT) 6.319 6.003 5.687 6.635 6.951Galactooligosaccharides (GOS) 17.67 17.67 17.67 17.67 17.67 Whey proteinconcentrate 12.20 12.20 12.20 12.20 12.20 Potassium citrate 1.277 1.2771.277 1.277 1.277 Calcium carbonate 996.1 g 996.1 g 996.1 g 996.1 g996.1 g Soy lecithin 685.0 g 685.0 g 685.0 g 685.0 g 685.0 gMonoglycerides 685.0 g 685.0 g 685.0 g 685.0 g 685.0 g ARA oil 684.2 g684.2 g 684.2 g 684.2 g 684.2 g Nucleotide/chloride premix 568.9 g 568.9g 568.9 g 568.9 g 568.9 g Potassium chloride 429.7 g 429.7 g 429.7 g429.7 g 429.7 g Ascorbic acid 293.8 g 293.8 g 293.8 g 293.8 g 293.8 gVitamin mineral premix 276.9 g 276.9 g 276.9 g 276.9 g 276.9 g DHA oil256.1 g 256.1 g 256.1 g 256.1 g 256.1 g Carrageenan 200.0 g 200.0 g200.0 g 200.0 g 200.0 g Magnesium chloride 173.3 g 173.3 g 173.3 g 173.3g 173.3 g Ferrous sulfate 112.7 g 112.7 g 112.7 g 112.7 g 112.7 gCholine chloride 104.8 g 104.8 g 104.8 g 104.8 g 104.8 g Vitamin A, D₃,E, K₁ premix 86.90 g 86.90 g 86.90 g 86.90 g 86.90 g Citric acid 57.50 g57.50 g 57.50 g 57.50 g 57.50 g Mixed carotenoid premix 41.90 g 41.90 g41.90 g 41.90 g 41.90 g Sodium chloride 23.50 g 23.50 g 23.50 g 23.50 g23.50 g L-carnitine 6.40 g 6.40 g 6.40 g 6.40 g 6.40 g Tricalciumphosphate AN AN AN AN AN Potassium phosphate monobasic AN AN AN AN ANPotassium hydroxide AN AN AN AN AN AN = as needed

Examples 26-30

Examples 26-30 illustrate human milk fortifier liquids of the presentdisclosure, the ingredients of which are listed in the table below. Allingredient amounts are listed as kilogram per 1000 kilogram batch ofproduct, unless otherwise specified.

Ingredient Ex. 26 Ex. 27 Ex. 28 Ex. 29 Ex. 30 Water Q.S. Q.S. Q.S. Q.S.Q.S. Non-fat milk 353 353 353 353 353 Corn Syrup Solids 85.3 85.3 85.385.3 85.3 Medium Chain Triglycerides 53.2 53.2 53.2 53.2 53.2 WheyProtein Concentrate 47.2 47.2 47.2 47.2 47.2 HMO Mixture 18.957 18.00917.061 19.905 20.853 6′sialyllactose (6′SL) 6.319 6.003 5.687 6.6356.951 2′fucosyllactose (2′FL) 6.319 6.003 5.687 6.635 6.951Lacto-N-neotetraose (LNnT) 6.319 6.003 5.687 6.635 6.951 CalciumPhosphate 25.5 25.5 25.5 25.5 25.5 Ascorbic Acid 5.6 5.6 5.6 5.6 5.6Potassium Citrate 3.1 3.1 3.1 3.1 3.1 Magnesium Chloride 2.8 2.8 2.8 2.82.8 Sodium Citrate 1.4 1.4 1.4 1.4 1.4 Sodium Chloride 1.4 1.4 1.4 1.41.4 Soy Lecithin 609 g 609 g 609 g 609 g 609 g M-Inositol 500 g 500 g500 g 500 g 500 g Niacinamide 400 g 400 g 400 g 400 g 400 g ARA Oil 313g 313 g 313 g 313 g 313 g Tocopherol Acetate 310 g 310 g 310 g 310 g 310g Zinc Sulfate 300 g 300 g 300 g 300 g 300 g Calcium Pantothenate 182 g182 g 182 g 182 g 182 g Ferrous Sulfate 133 g 133 g 133 g 133 g 133 gDHA Oil 116 g 116 g 116 g 116 g 116 g Vitamin A Palmitate 100 g 100 g100 g 100 g 100 g Cupric Sulfate 51.0 g 51.0 g 51.0 g 51.0 g 51.0 gThiamine Hydrochloride 50.0 g 50.0 g 50.0 g 50.0 g 50.0 g Riboflavin47.0 g 47.0 g 47.0 g 47.0 g 47.0 g Pyridoxine Hydrochloride 27.0 g 27.0g 27.0 g 27.0 g 27.0 g Vitamin D3 20.0 g 20.0 g 20.0 g 20.0 g 20.0 gFolic Acid 3.5 g 3.5 g 3.5 g 3.5 g 3.5 g Biotin 3.4 g 3.4 g 3.4 g 3.4 g3.4 g Manganous Sulfate 1.5 g 1.5 g 1.5 g 1.5 g 1.5 g Phylloquinone 1.2g 1.2 g 1.2 g 1.2 g 1.2 g Cyanocobalamin 100 mg 100 mg 100 mg 100 mg 100mg Sodium Selenate 43.0 mg 43.0 mg 43.0 mg 43.0 mg 43.0 mg

Examples 31-35

Examples 31-35 illustrate spray dried nutritional powders of the presentdisclosure, the ingredients of which are listed in the table below. Allingredient amounts are listed as kilogram per 1000 kilogram batch ofproduct, unless otherwise specified.

Ingredient Ex. 31 Ex. 32 Ex. 33 Ex. 34 Ex. 35 Condensed Skim Milk 698.5698.5 698.5 698.5 698.5 Lactose 386.0 386.0 386.0 386.0 386.0 High oleicsafflower oil 114.4 114.4 114.4 114.4 114.4 Soybean oil 85.51 85.5185.51 85.51 85.51 Coconut oil 78.76 78.76 78.76 78.76 78.763′sialylallactose (3′SL) 0.3792 0.3604 0.3412 37.916 36.0188Galactooligosaccharides (GOS) 69.50 69.50 69.50 69.50 69.50 Whey proteinconcentrate 51.08 51.08 51.08 51.08 51.08 Potassium citrate 9.168 9.1689.168 9.168 9.168 Calcium carbonate 4.054 4.054 4.054 4.054 4.054 Soylecithin 1.120 1.120 1.120 1.120 1.120 ARA oil 2.949 2.949 2.949 2.9492.949 Nucleotide/chloride premix 2.347 2.347 2.347 2.347 2.347 Potassiumchloride 1.295 1.295 1.295 1.295 1.295 Ascorbic acid 1.275 1.275 1.2751.275 1.275 Vitamin mineral premix 1.116 1.116 1.116 1.116 1.116 DHA oil1.113 1.113 1.113 1.113 1.113 Magnesium chloride 1.038 1.038 1.038 1.0381.038 Sodium chloride 579.4 g 579.4 g 579.4 g 579.4 g 579.4 g Ferroussulfate 453.6 g 453.6 g 453.6 g 453.6 g 453.6 g Choline chloride 432.1 g432.1 g 432.1 g 432.1 g 432.1 g Vitamin A, D₃, E, K₁ premix 377.2 g377.2 g 377.2 g 377.2 g 377.2 g Ascorbyl Palmitate 361.3 g 361.3 g 361.3g 361.3 g 361.3 g Mixed carotenoid premix 350.1 g 350.1 g 350.1 g 350.1g 350.1 g Mixed Tocopherols 159.2 g 159.2 g 159.2 g 159.2 g 159.2 gL-carnitine 26.30 g 26.30 g 26.30 g 26.30 g 26.30 g Riboflavin 3.181 g3.181 g 3.181 g 3.181 g 3.181 g Tricalcium phosphate 0-5.23 0-5.230-5.23 0-5.23 0-5.23 Potassium phosphate monobasic 0-5.23 0-5.23 0-5.230-5.23 0-5.23 Potassium hydroxide AN AN AN AN AN AN = as needed

Example 36

In this Example, the effect of purified human milk oligosaccharides(HMO) on in vitro inhibition of viral infectivity is analyzed.

Samples are prepared by co-incubation of a uniform virus dose of fromabout 500 units/mL to about 1,000 units/mL of one of three respiratoryviruses: (1) respiratory syncytial virus (RSV); (2) human parainfluenzavirus (HPIV3); or (3) H1N1 influenza virus with one of the followingHMOs: (1) 3′-sialyllactose (3′SL); (2) 6′-sialyllactose (6′SL); (3)3′-fucosyllactose (3′FL); (4) 2′-fucosyllactose (2′FL); (5)lacto-N-neotetraose (LNnT); or (6) sialic acid (SA). The HMOs are addedat concentrations of either 1 mg/mL or 10 mg/mL. The antiviralactivities of the various HMOs on the respiratory viruses are evaluated,and the results are shown in the table below:

IC50 (mg HMO/mL) HMO RSV HPIV3 H1N1 Influenza 3′SL >10 >10 ~56′SL >10 >10 ~10 3′FL ~5 ~2 ~5 2′FL >10 >10 ~10 LNnT >10 NT >10 SA NT ~2~5 NT = Not Tested

The results show that 3′FL, at a concentration of 1 mg/ML (IC50 ˜2-5mg/ML), has anti-viral activity for all three respiratory viruses. Thisresult is unexpected as previous published reports show only sialylatedoligo forms providing antiviral activity. SA significantly inhibitsHPIV3 and H1N1 viruses at a concentration of 1 mg/mL. H1N1 influenzavirus is also inhibited by 3′SL at a concentration of 1 mg/mL.

Example 37

In this Example, the ability of various HMOs to block H1N1 influenzavirus infectivity in vitro is analyzed.

Virus infectivity is assessed by observing cytopathic effect (CPE) andquantifying virus focus forming units. To create virus stocks, H1N1influenza virus is purchased from ATCC (VR 1469) and expanded inMadin-Darby Canine Kidney (MDCK) epithelial cells (ATCC CCL-34).Cell-free supernatants are frozen in aliquots to maintain stock virus.During initial virus culture and expansion to create virus stocks, cellCPE is observed.

To quantify virus infectivity, an immunocytochemical focus forming unit(FFU) assay is developed using commercially purchased mouse monoclonalantibodies against the virus nucleoprotein coupled with a biotinylatedanti-mouse IgG secondary antibody. To visualize virus-infected cellfoci, color development is performed using Strepavidin HRP (ABC fromVector Laboratories, Inc.). Although the total number of virus fociappear proportional to the infecting virus concentration, the foci arequite large, disperse, and there are numerous individually infectedcells that do not form foci, especially at higher virus concentrations.As this makes quantifying of virus infectivity difficult andtime-consuming, the FFU assay is further refined by varying virusconcentration and by applying an overlay medium of Tragacanth gum tohelp reduce Brownian movement spread of the virus throughout the celllayer.

The use of Tragacanth gum improves the assay by reducing the number ofindividually infected cells while still allowing for the formation ofreadily observable foci. While the foci vary in size, with some beingquite large, they are still easily quantified and directly proportionalto virus concentration or titer by using a grid technique during theenumeration.

Once verified, the assay is used with various HMOs for the ability toblock H1N1 virus infectivity. Specifically, the HMOs are added, atconcentrations of 0.01 mg/mL, 0.1 mg/mL, 1.0 mg/mL, and 10 mg/mL, to theinoculating virus suspension, incubated at 37° C. for one hour, and thenadded to MDCK monolayer cells. This mixture is allowed to bind to thecell layer for thirty minutes at 37° C. The cell layer is then washed,and the cells are further incubated for approximately 18-24 hours beforefixing and processing for immunocytochemical staining. The results areshown in FIG. 1.

As shown in FIG. 1, 3′FL, 3′SL, and SA each inhibit virus infectivity bygreater than 90% when used at a concentration of 10 mg/mL. 2′FL and 6′SLinhibit infectivity by approximately 60% at 10 mg/mL.

Example 38

In this Example, nutritional compositions including various HMOs areevaluated for their effects on reducing oxidative stress in pretermpiglets.

Preterm piglets are harvested by caesarian section (CS) at 92% ofgestation. Piglets receive total parenteral nutrition (TPN) for 48hours. After 48 hours, TPN is ceased and the piglets are randomized intothree groups: a formula group (n=7) that is fed Enfamil® Lacto-Free,commercially available from Mead Johnson, Evansville, Ind.; a treatmentgroup (n=9) that is fed Enfamil® Lacto-Free with the addition of acombination of 400 mg/L 6′SL, 1500 mg/L 2′FL, and 200 mg/L LNnT; and acolostrum group (n=5) that is fed bovine colostrum. Piglets are fedtheir respective feeding enterally at a rate of 120 mL formula per kgbody weight for the next 48 hours. Piglets are then euthanized after 48hours of enteral nutrition (EN), or earlier if a piglet develops signsof necrotizing enterocolitis. Blood is collected via an umbilical arterycatheter, and plasma is separated from the blood and stored at −70° C.until analyzed.

Glutathione (GSH) concentrations are measured in plasma taken from thepiglets just prior to feeding time (time 0), and at 6 hours, 12 hours,24 hours, 36 hours, and 48 hours after feeding using a commerciallyavailable assay (NWLSS Glutathione Assay #NWK-GSH01, Northwest LifeScience Specialties, Vancouver, Wash.). The results are shown in FIG. 2.

As shown in FIG. 2, the concentration of GSH in blood plasma from thecontrol group declines from time 0 to 6 hours after feeding. GSH remainslower in the control group 24 hours after EN. In contrast, piglets fedthe composition with a combination of HMOs have a pattern of bloodplasma GSH levels that are comparable to the colostrum piglets.

Example 39

In this Example, the abilities of 3′SL, 6′SL, and LNnT to reducevirus-induced inflammation in vitro are demonstrated.

Specifically, either 3′SL or 6′SL is added, at concentrations of 0.1mg/mL, 0.2 mg/mL, or 0.5 mg/mL to fresh peripheral blood mononuclearcells (PBMCs) and incubated at 37° C. in 5% CO₂ to pretreat the cellsfor approximately 24 hours. LNnT is added, at concentrations of 0.1mg/mL, 0.2 mg/mL, or 1 mg/mL to PBMCs and incubated at 37° C. in 5% CO₂to pretreat the cells for approximately 24 hours. Lactose is included asa carbohydrate control. Matched endotoxin unit concentration controlsare included to allow differentiation of ingredient effects frominherent low levels of endotoxin. Some variables are then incubated withRSV at a multiplicity of infection (MOI) of 0.1 for approximately 1 hourat 37° C. in 5% in CO₂. Uninfected control variables are incubated withmedium for approximately 1 hour at 37° C. in 5% CO₂. After approximatelyone hour, fresh medium alone, or fresh medium containing the appropriateconcentration of 3′SL, 6′SL, LNnT, lactose, or endotoxin is added to theappropriate tubes and the cells are incubated for 48 hours at 37° C. in5% CO₂. Supernatants are collected at 24 and 48 hours post-infection.Cytokines are measured in supernatants for each variable at 24 and 48hours to assess the effects of HMOs on the early immune response to RSV.Cytokines are measured using custom Bio-Plex Human cytokine kits fromBio-Rad.

IP-10 is a CXC chemokine that attracts, binds to and activates the CXCR3receptor on natural killer cells and memory T cells. IP-10 is expressedby monocytes and a number of other cells, and is induced by interferon.A positive correlation exists between RSV clinical disease severity inchildren (as measured by: length of hospital stay, fever, and number ofdays supplemental O₂ is required) and serum IP-10. Therefore, a decreasein IP-10 signals a decrease in severity of RSV disease experienced.

IP-10 results for 3′SL and 6′SL are detailed in FIGS. 3 and 4 which showthat 6′SL downregulates IP-10 in virus-infected variables in both donorstested. Note that 6′SL is able to reduce IP-10 to levels found inuninfected controls. 3′SL is not effective in Donor B, but downregulatesRSV-induced IP-10 in Donor E. These data show both 3′SL and 6′SL dampenRSV-induced IP-10, but that 6′SL is more effective at downregulation ofIP-10. Results also suggest that levels below 0.1 mg/mL of 6′SL as wellas levels greater than 0.5 mg/mL may be effective at reducing IP-10 insome individuals.

IP-10 results for LNnT are detailed in FIGS. 5 and 6 and show somevariability in donor response, but surprisingly, LNnT clearlydownregulates IP-10 in virus-infected variables in both donors. Notethat LNnT is able to reduce IP-10 to levels found in uninfectedcontrols. Results also suggest that levels between 0.2 and 1 mg LNnT/mLas well as greater than 1 mg/mL may be effective at reducing IP-10 insome individuals. Inclusion of matched endotoxin unit concentrationcontrols clearly indicates that the decrease in IP-10 is not due to thepresence of very low levels of endotoxin in the LNnT.

In FIGS. 7 and 8, cytokine results also surprisingly show 6′SL increasesinterleukin 10 (IL-10) concentration in a dose-dependent manner in thepresence or absence of RSV. IL-10 results for LNnT are shown in FIGS. 9and 10. Surprisingly, LNnT increases IL-10 concentration in adose-dependent manner in the presence or absence of RSV. IL-10 isproduced by activated CD8+ T-cells, by CD4+ T-cells after bothantigen-specific and polyclonal activation, and by monocytes followingcell activation by bacterial lipopolysaccharides. Inclusion of matchedendotoxin unit concentration controls clearly differentiates that theincrease in IL-10 is not due to the presence of very low levels ofendotoxin in the 6′SL or the LNnT.

Surprisingly, it is found that pretreatment for 24 hours by 6′SL, 3′SL,or LNnT is effective in reducing inflammation caused by RSV. Moreover,6′SL and LNnT are shown to be more effective than 3′SL at dampeningvirus-induced inflammation as measured by a decrease in IP-10. Further,it is shown that 6′SL is immunomodulatory in the absence of the virus,as the inclusion of 6′SL induces and/or modifies the production ofmonocyte-derived cytokines such as IL-10, MIP-1β, Interferon-γ, IL-8,IL-1α, IL-1β, and IL-1ra. Surprisingly, 3′SL is also immunomodulatory inthe presence or absence of the virus, as the inclusion of 3′SL inducesand/or modifies the production of monocyte-derived cytokines such asMIP-1β, Interferon-γ, IL-8, and IL-1ra. Surprisingly, LNnT is alsoimmunomodulatory in the presence or absence of the virus, as theinclusion of LNnT induces and/or modifies the production ofmonocyte-derived cytokines such as IL-10, MIP-1β, Interferon-γ, IL-8,IL-1α, IL-1β, and IL-1ra.

Example 40

In this example, the ability of 2′FL to reduce extracellular reactiveoxygen species (ROS) in both uninfected and RSV infected peripheralblood mononuclear cells (PBMCs) in the presence or absence of phorbalmyristate acetate (PMA) is demonstrated.

On day 0, fresh human PBMCs are isolated from whole blood. 2′FL is addedto PBMCs at concentrations of 0.1 mg/mL, 0.2 mg/mL, or 1 mg/mL andincubated for approximately 24 hours at 37° C. in 5% CO₂. On day 1, thecell supernatants are removed and the PBMCs are incubated with mediumalone or medium plus Respiratory Syncytial Virus (RSV) for approximately1 hour at 37° C. in 5% CO₂ at a multiplicity of infection (MOI) of 1.After approximately 1 hour, fresh medium alone or containing theappropriate concentration of 2′FL is added to the appropriate tubes, andthe cells are incubated for 48 hours at 37° C. in 5% CO₂.

On day 3, 20% (3.5×105 cells) of the PBMC culture is removed foranalysis of NADPH oxidase derived reactive oxygen production using thechemiluminescent probe isoluminol. The isolated cells are divided evenlyinto two tubes with the reaction mixture containing isoluminol andhorseradish peroxidase. One tube is treated with phorbol 12-myristate13-acetate to induce activation of the NADPH oxidase enzyme complex.Reactive oxygen derived chemiluminescence is monitored in a luminometerover a 60 minute period. Reactive oxygen production is quantified byintegrating the area under the curve over the 60 minute period. Theremaining PBMC supernatants and cell lysates are collected separately,aliquotted and stored frozen at −70° C. for later analysis.

The results as shown in FIG. 11B indicate that 2′FL dampens release ofextracellular reactive oxygen species (ROS) in uninfected (0.1-0.2 mg2′FL/mL) and infected cells (0.1-1.0 mg 2′FL/mL) in the absence ofphorbol myristate acetate (PMA). Further, the results as shown in FIG.11A indicate that 2′FL dampens ROS in uninfected (0.1-1.0 mg 2′FL/mL)and infected (0.1-0.2 mg 2′FL/mL) PBMCs in the presence of PMA.Dampening ROS may decrease RSV induced oxidative stress damage to lungsand other tissues.

What is claimed is:
 1. A method of reducing the incidence ofnon-pathogenic oxidative stress in an infant, toddler, or child, themethod comprising administering to the infant, toddler, or child anutritional composition comprising an effective amount of a human milkoligosaccharide selected from 2′-fucosyllactose and 6′-sialyllactose andat least one carotenoid; wherein when the human milk oligosaccharidecomprises 6′-sialyllactose, the 6′-sialylactose is present in an amountof from 0.001 mg/mL to 2 mg/mL.
 2. The method of claim 1, wherein thenutritional composition further comprises vitamin C.
 3. The method ofclaim 1, wherein the nutritional composition comprises from about 0.001mg/mL to about 20 mg/mL of total human milk oligosaccharides.
 4. Themethod of claim 1, wherein the nutritional composition comprises from0.001 mg/mL to less than 2 mg/mL of 2′-fucosyllactose.
 5. The method ofclaim 1, wherein the composition comprises from 0.001 mg/mL to less than2 mg/mL lacto-N-neotetraose.
 6. The method of claim 1, wherein thenutritional composition further comprises at least one of a prebiotic, aprobiotic, a long chain polyunsaturated fatty acid, a short chain fattyacid, an antioxidant, a protein, vitamins, and minerals.
 7. The methodof claim 1, wherein the carotenoid is selected from the group consistingof lutein, lycopene, and beta-carotene.
 8. The method of claim 7,wherein the composition comprises the carotenoid in a total amount offrom about 0.001 μg/mL to about 10 μg/mL.
 9. A method of reducing theincidence of non-pathogenic oxidative stress in an infant, toddler, orchild, the method comprising administering to the infant, toddler, orchild a nutritional composition comprising an effective amount of6′-sialyllactose, 2′-fucosyllactose, and lacto-N-neotetraose; whereinthe 6′-sialylactose is present in an amount of from 0.001 mg/mL to 2mg/mL.
 10. The method of claim 9, wherein the composition furthercomprises vitamin C.
 11. The method of claim 9, wherein the6′-sialyllactose, 2′-fucosyllactose, and lacto-N-neotetraose are presentin a total amount of from about 0.001 mg/mL to about 20 mg/mL.
 12. Themethod of claim 9, wherein the composition comprises from 0.001 mg/mL toless than 2 mg/mL of 2′-fucosyllactose.
 13. The method of claim 9,wherein the composition comprises from 0.001 mg/mL to less than 2 mg/mLof lacto-N-neotetraose.
 14. The method of claim 9, wherein thecomposition further comprises at least one of a prebiotic, a probiotic,a long chain polyunsaturated fatty acid, a short chain fatty acid, anantioxidant, a milk protein of human origin, a milk protein of bovineorigin, a protein, vitamins, and minerals.
 15. The method of claim 9,wherein the composition further comprises a carotenoid selected from thegroup consisting of lutein, lycopene, and beta-carotene.
 16. The methodof claim 15, wherein the composition comprises the carotenoid in a totalamount of from about 0.001 μg/mL to about 10 μg/mL.
 17. A method ofreducing tissue damage due to non-pathogenic oxidative stress in aninfant, toddler, or child, the method comprising administering to theinfant, toddler, or child a nutritional composition comprising aneffective amount of a human milk oligosaccharide selected from2′-fucosyllactose and 6′-sialyllactose and at least one carotenoid;wherein when the human milk oligosaccharide comprises 6′-sialylactose,the 6′-sialylactose is present in an amount of from 0.001 mg/mL to 2mg/mL.